Protein-protein interactions play a central role in cellular signaling. The frequency and strength of protein interactions depend on the local concentrations of two proteins and their affinity for one another. Monitoring and modulating protein interactions provides important mechanistic insight into cellular processes and is essential for developing pharmacological intervention in disease states. Current investigation of protein interactions in-vitro and in live cells commonly employ fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins to extract information about inter- and intramolecular changes in proteins. However, this approach to understand cell signaling is seriously impeded by two major obstacles: (1) Within a live cell, no methods exist to systematically vary the local concentration of interacting proteins in order to translate interaction into cellular function. (2) Conventional FRET detection suffers severely from low signal-to-noise-ratio (SNR) due to the very small changes occurring in donor and acceptor emission during protein interactions, and the strong pre-existing donor and acceptor emission background. We propose to develop an optofluidic FRET laser system that synergizes two distinct, emerging technologies, systematic protein affinity strength modulation (SPASM) and the optofluidic laser, for unprecedented capability of analyzing protein interactions in-vitro and in live cells in a controllable manner. SPASM relies on a modular and tunable ER/K a-helix to link two interacting proteins. The ER/K a-helix can be engineered to systematically change the protein interaction frequency. Meanwhile, the optofluidic laser acts as a highly sensitive FRET detector to provide a quantitative readout. It employs stimulated laser emission as the sensing signal. When FRET takes place inside the laser cavity, a small change in FRET induced by protein interactions will be optically amplified by the optofluidic laser, thus resulting in a drastic increase in the FRET signal. In addition, due to the unique optical design of the optofluidic laser, the pre-existing donor and acceptor emission background can virtually be eliminated. Therefore, orders of magnitude improvement in FRET sensitivity can be obtained. Here, we will first systematically investigate the optofluidic FRET laser using ER/K a-helix modulated protein FRET pairs and benchmark our technology against conventional FRET detection. Then, we will use the optofluidic laser to study the ER/K a-helix modulated calmodulin (CAM)-peptide system in-vitro and in live cells, whose interaction can be varied widely by Ca2+ concentration. Finally, we will apply the optofluidic FRET laser to substantially enhance (>100 fold) the sensitivity of a live cell G-protein coupled receptor (GPCR) activation sensor developed using the SPASM technique. We have three specific aims: Aim 1: Investigate and benchmark the optofluidic FRET laser with ER/K a-helix modulated protein FRET pairs; Aim 2: Investigate and benchmark the optofluidic FRET laser with CAM-peptide in-vitro and in live cells; Aim 3: Substantially enhance (>100 fold) the sensitivity of a live cell GPCR activation sensor.